Figure 4

Importance of PI4P resynthesis by PI4KA on agonist-induced PIP2 depletion and receptor internalization. (A) PIP2 depletion efficiency was measured by BRET as detailed earlier in Fig. 1B, using the TSTS/4A mutant AT1R-FRB and applying a pretreatment for 10 min with either vehicle (left graph) or 10 nM GSK-A1 (A1), a specific inhibitor of PI4KA (right graph). Lipid depletion was triggered by AT1R stimulation (100 nM Ang II, red curves) at time point 0, followed by ionomycin (10 μM) for complete degradation of PIP2. The signal change was normalized to the first data point (100%) and the value of Renilla luciferase alone (0%). Black curves show unstimulated samples. Data show the mean ± SEM of 3 independent experiments, each performed in triplicate. (B) Internalization of β2AR was measured by BRET as detailed in Fig. 2A, with the distinction that the TSTS/4A mutant AT1R-FRB was expressed in all cells. Cells were pretreated with either vehicle (left panel) or A1 (10 nM, right panel) for 10 min. PIP2 depletion was evoked by activating AT1R-FRB at time point 0 with Ang II (100 nM, red curves). Black curves show data where cells were not treated with Ang II. After 5 min, β2AR-Sluc was stimulated with isoprenaline (1 μM). BRET ratio change was calculated by subtracting baselines not stimulated with isoprenaline. Data show the mean ± SEM of 3 independent experiments, each performed in triplicate. (C) The bar graph shows the rate of β2AR-Sluc internalization 10–15 min after isoprenaline (1 μM) treatment normalized to that in the nonpretreated and non-PIP2-depleted samples (clear gray bar). Data are the means (± SEM) of the last 5 measurement points of the curves from panel B. Data were analyzed using two-way ANOVA, which showed a significant difference (p < 0.01) between the groups and a significant interaction between Ang II and A1 treatments (p < 0.05). Bonferroni's post hoc test was used for pairwise comparisons, and only the A1-pretreated Ang II-stimulated sample was found to be significantly different from all others (p < 0.01). n = 3. (D) Internalization of β2AR was visualized by confocal microscopy. β2AR-Venus and AT1R TSTS/4A-mRFP were expressed in HEK 293A cells pretreated with vehicle (upper images) or A1 (10 nM, 10 min, lower images). PIP2 depletion was induced by activating AT1R with Ang II (100 nM, 5 min, right images only), and then β2AR was stimulated in all cells with isoprenaline (1 μM) for 15 min. Cells were then fixed, and confocal images of β2AR-Venus were taken. Representative images from three independent experiments. Scale bar: 15 μm.